Centrifuges are widely used to separate spermatozoa from semen and also for separating other types of viable cells, such as blood cells, bacteria and other microorganisms. There are generally three different types of centrifuges which are broadly classified as low-speed, high-speed and ultra-speed centrifuges. Low-speed centrifuges typically have a maximum rotor speed of less than 10,000 rpm and are used to harvest intact or viable cells. However, it is well known that even low-speed centrifuges can have adverse effects upon the quality of the harvested biological material due to the excessive centrifugal forces exerted upon the material.
Several authors have reported on the particularly poor results which commonly occur in the recovery of spermatozoa from centrifugation. Van der Ven H. H., Jeyendran R. S., Tunnerhoff A., Hoebbel K., Al-Hasani S., Diedrich K., Krebs D., and Perez-Pelaez M., Glass Wool Column Filtration of Human Semen: Relationship Swim Up Procedure and IVF Outcome, Human Reprod. 1988; 3:85-8; Rhemrev J., Jeyendran R. S., Vermeiden J. P., and Zaneveld L. J. D., Human Sperm Selection by Glass Wool Filtration and a Two-Layer Discontinuous Percoll Gradient Centrifugation, Fertil. Steril. 1989; 51:685-90. It is believed that such poor results are due to the standard practice of continuously centrifuging semen or other samples for a period of time at high centrifugal forces which can adversely effect the quality of the recovered cells. Lower centrifugal forces are sometimes used but require an increase in the centrifugation time in order to achieve an effective rate of sedimentation and a satisfactory concentration of viable cells in the recovered samples. However, increasing the centrifugation time has adverse effect upon the quality of recovered samples similar to the results achieved when applying high centrifugal forces. In fact, the deleterious effects of such centrifugation have been attributed to intracellular and ultrastructural damage to spermatozoa. Makler A., Murillo O., Huszar G., Tarlatzis B., De Cherney A., and Nabtolin F., Improved Technique for Separating Motile Spermatozoa From Human Semen. II An Autraumatic Centrifugation Method, Int. J. Androl. 1984; 7:71-8; Mack S. R. and Zaneveld L. J. D., Acrosomal Enzymes and Ultrastructure of Unfrozen and Cryotreated Human Spermatozoa, Gamete Res. 1987; 18:375-83; Makler A. and Jakobi P., Effects of Shaking and Centrifugation on Human Sperm Motility, Arch. Androl. 1981; 7:21-6. Such adverse effects reduce sperm motility and result in poor quality specimens for analysis and artificial insemination.
In a typical prior art method of centrifuging semen, the semen sample is continuously centrifuged at a constant relative centrifugal force of between 200 and 1000 g for a time period of 5 to 30 minutes. When the g force is high (1000 g), the time period tends to be shorter (5 minutes), and when the g rate is low (200 g), the time period tends to be higher (20 to 30 minutes). In both cases, the prior art methods require either a high centrifugal force or a long centrifugation period in order to achieve an effective rate of sedimentation and to recover a sample having a high sperm concentration. However, since high centrifugal forces and long centrifugation periods are the primary factors which adversely affect viable cells, both methods have adverse effects on the quality of the recovered cells.
An important aspect of this invention therefore lies in the discovery of pulsing and oscillating methods of centrifuging which yield a higher number of viable cells than the prior art methods. The inventive methods employ either pulsing or oscillating the relative centrifugal force applied to the samples to gradually and gently nudge or urge the viable cells towards the bottom of the centrifugation chambers. By either pulsing or oscillating the relative centrifugal force, the inventive methods avoid exerting a continuous centrifugal force on the viable cell samples such as occurs in the prior art methods.
Briefly, the pulsing method comprises first obtaining a sample of viable cells and adding that sample to a biologically compatible medium. The sample and medium are then placed in the chamber of a centrifuge. The chamber is then rotated so that a predetermined centrifugal force is applied to the sample and the rotation of the chamber is then stopped so that the chamber comes to rest. Thereafter, the rotating and stopping steps are alternately repeated until at least three total rotating steps have been performed. Preferably, the total time of the rotating steps does not exceed approximately 5 minutes. The samples are then removed from the centrifuge. It has been found that such a pulsing method yields a significantly higher number of viable cells with higher quality than the prior art centrifuging methods.
During the rotating step, the predetermined centrifugal force applied to the sample is about 200 to 1000 g, preferably about 1000 g. Each rotating step has a time period of approximately 30 seconds to 2 minutes, and each of the stopping steps between the rotating steps also has a time period of approximately 30 seconds to 2 minute. In a preferred embodiment, each of the rotating steps and each of the stopping steps has a time period of approximately 1 minute. The pulsing method may be used to centrifuge a variety of different types of viable cells such as blood cells, various microorganisms, spermatozoa, bacteria, etc.
The oscillating method is similar to the pulsing method except that the centrifuge is not completely stopped. Instead, the centrifugal force applied to the sample is oscillated between a first centrifugal force in one rotating step and a second centrifugal force in a second rotating step. The first centrifugal force is higher than the second centrifugal force and the periods of operation at the lower centrifugal force interrupt the periods of operation at the higher centrifugal force to avoid the continuous application of a high centrifugal force to the viable cell samples. The rotation of the chamber at the first and second centrifugal forces is alternately repeated until at least three total rotating steps at the higher centrifugal force have been completed. Preferably, the total time of rotation of the chamber and samples does not exceed approximately 5 minutes.
During the first and second rotating steps, the first centrifugal force is about 750 to 1000 g and the second centrifugal force is about 200 to 750 g, respectively. In one embodiment, the first centrifugal force was about 1000 g and the second centrifugal force was about 500 g. The time period for operation at the first and second centrifugal forces can vary between a period of 10 seconds and 1 minute and the total time of rotation generally should not exceed approximately 5 minutes. While in some circumstances continued rotation beyond a time period of 5 minutes might result in increased sedimentation of a sample, it is believed more likely that the increased rotation time may damage the viable cells and that is why the preferred rotation time does not exceed approximately 5 minutes. It has been found that such an oscillating method yields a significantly higher number of viable cells with higher quality than the standard centrifugation method of applying a continuous centrifugal force to the viable cell samples.
Other objects, features, and advantages of the present invention will become apparent from the following specification and drawings.